The dengue virus (DENV) is an increasing threat to global public health, but there is no known vaccine or treatment for infection. As the most rapidly spreading mosquito-borne virus worldwide, there is an urgent need to train biomedical scientists in dengue virus biology. Development of a protein-targeting vaccine to prevent infection has proven extremely difficult, suggesting that non-protein targets may prove more useful in therapeutic development. The RNA structures in the viral genome essential for dengue replication are potential targets, but we currently have only a limited knowledge of a few DENV RNA structures and their functions, and this existing information is largely confined to the 5' and 3' untranslated regions (UTRs). The goal of this research and training program is to determine the role of RNA structures in the dengue virus genome during the viral replication cycle; this knowledge will both advance our general understanding of dengue viral biology and will contribute to the development of novel therapeutic interventions. To accomplish this goal, the primary objective of this research is to experimentally characterize the secondary structure of an authentic ex virio viral RNA genome at nucleotide resolution using a chemical mapping technology called SHAPE developed in the sponsor's laboratory and to examine the biological roles of key selected structures. The expected outcome of this study is the construction of a comprehensive secondary structure map of a DENV RNA, identification of new RNA structure-function relationships, characterization of principles of mosquito-based RNAi response against dengue infection, and advanced training of the applicant in next-generation structure- function analyses of viral replication mechanisms. Ultimately, this research will lead to the identification of novel anti-dengue drug targets that motivate future therapeutic studies. The studies proposed herein will also provide the applicant with training in virology, RNA biology, bioinformatics, high throughput data analysis, and structure characterization of large RNAs, all of which are areas pertinent to the applicant's long-term focus on becoming a leader in the field of RNA-based diseases. PUBLIC HEALTH RELEVANCE: The dengue virus is a substantial and increasing threat to global public health with no known vaccine or treatment for infection; to develop anti-dengue therapeutics and prevent virus transmission, we need to better understand dengue virus biology and train biomedical scientists in this increasingly important field. To this end, the goal of this program is to train the applicant as a new investigator in dengue biology and in high-throughput RNA structure-determination technologies with the ultimate goal of experimentally defining the structure of an entire dengue RNA genome. The proposed experiments will reveal novel RNA structure-function relationships, establish principles of mosquito RNAi response against dengue infection, and identify multiple anti-dengue drug targets that can be pursued in future therapeutic studies.